There are few published protocols for the transformation and regeneration of Allium species. The Allium crop species are probably the most economically important vegetable species for which transformation technology is still difficult. For other major vegetable crops, efficient transformation systems have been produced.
Initially, many monocotyledons were thought to be unsusceptible to Agrobacterium-mediated transformation. The development of direct gene transfer techniques soon led to bombardment being the favoured method of monocotyledon transformation. However, direct gene transfer is not without its problems. Often, low transformation frequencies and a high frequency of unusual integration patterns has been observed in transgenic plants. Recently, Agrobacterium-mediated transformation of monocotyledons has gained favour and many monocotyledonous species (including rice, wheat, barley, maize and sugarcane) have now been transformed using this method. A key component in the success of these systems has been the use of highly embryogenic tissue types, and precise post transformation selection protocols.
Recently, Haseloff (1997) has modified the gfp gene to enhance its use as a transgenic marker gene in viable plant systems. Green fluorescent protein (GFP) enables researchers to follow precisely the fate of any cells expressing this gene and so optimise post transformation cell survival. Such a system has been useful in the development of the allium transformation protocol reported here.
As monocotyledons, the Allium species were predisposed to be recalcitrant to transformation. Onions (Allium cepa L) are a crop with diverse environmental requirements. It has, therefore, been relatively understudied with respect to the application of biotechnology. There are only a few reports of DNA delivery to Alliums (Klein 1987; Dommisse et al. 1990; Eady et al. 1996; Barandiaran et al. 1998). Three workers used direct gene transfer whilst Dommisse et al. (1990) demonstrated that Agrobacterium-mediated transformation may be possible. Some reports of regeneration protocols for Alliums that are appropriate for transformation study have become available (Hong and Deberg 1995; Xue et al. 1997; Eady et al. 1998; Saker 1998). Only one report exists on the development of potential selective agents for use in Allium transformation (Eady and Lister 1998a).
More recently U.S. Pat. No. 7,112,720 describes a method of transforming onion plants using Agrobacterium mediated transformation and using immature embryos as the explant source. Since then, there have been several further reports of transformation of onion (Zheng et al. 2001b; Baster et al. 2003; Aswath et al. 2006), leek (Eady et al. 2005) and garlic (Kondo et al. 2000; Eady et al. 2005). However, all of these reports are inefficient and based around immature embryo use (e.g. all previous Eady et al. transformations) or the use of floral tissue (Bastar et al 2003) or callus derived from seedling radicle tissue (Aswath et al 2006) or mature zygotic embryo callus (Zheng et al 2001), or callus derived from meristamatic shoot primordia (Kondo et al 2000). None of the protocols thus far developed have used direct regeneration from immature leaf tissue as outlined in this invention.
To date leaf tissue has not been used as, or considered a viable, source of tissue for Allium transformation. Leaf tissue has not been investigated or considered a useful starting material due to difficulties in getting good transformation of leaf tissue. Furthermore, leaf cells from monocotyledons are notoriously recalcitrant to regeneration, unlike dicotyledonous leaf tissue which is often very easily regenerated. There are no reports of successful direct regeneration from isolated allium leaf tissue and only low frequency regeneration from twin scales, in which a few cells on the abdaxial side of the upper leaf in the twin scale regenerates (reviewed by Eady 1995 updated in Eady and Hunger 2008). There has been one recent report of regeneration from garlic leaf via callus and somatic embryogenesis (Fereol et al. 2002). The identification in this work of ‘plastic’ immature leaf cell types from the leaf base of allium species and their use for transformation and regeneration is an unexpected and novel finding that can greatly enhance the application of biotechnology to crop allium species.